PD-1H (VISTA) Induces Immune Evasion in Acute Myeloid Leukemia

Despite progress in understanding the molecular pathogenesis in acute myeloid leukemia (AML), the disease remains highly fatal with limited therapeutic options. There is a need for novel approaches for management of AML that can overcome resistant disease. Immune checkpoint inhibitor therapy has emerged as a successful treatment strategy in several solid tumors and refractory Hodgkin lymphoma. However, preliminary data from clinical trials to date indicate that anti-PD-1 antibodies have limited single-agent activity in AML. This suggests that co-inhibitory molecules other than PD-1 may induce immune evasion in AML. Programmed Death-1 Homolog (PD-1H, VISTA) is a novel co-inhibitory molecule that induces immune evasion in solid tumors. Interestingly, The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia database both reveal that PD-1H is over-expressed in AML at the transcript level. While we found that myeloid subsets and T cells have the expression of PD-1H in bone marrow of healthy donors, we demonstrated that PD-1H protein is highly expressed in bone marrow of 8 (of 10) human AML donors using immunohistochemistry. To determine whether the high expression of PD-1H in AML bone marrow is associated with the expression of PD-1H in AML blasts, flow cytometric analyses were performed in bone marrow aspirate of human CD34+ AML donors. Mean Fluorescent Intensity (MFI) of CD34+ AML blasts was significantly higher when compared to normal CD34+ progenitor cells (MFI: 257±82 (AML CD34+ blasts) vs 10±4 (normal CD34+ progenitor cells), N=6, p<0.05). To test our hypothesis that PD-1H induces immune evasion in AML, we transplanted PD-1H-expressing murine myeloid leukemia cells (C1498) into syngeneic PD-1H knockout (KO) or wild type (WT) littermate mice. Whole body bioluminescence (unit: radiance) was used to determine the level of luciferase activity reflecting in vivo growth of luciferase expressing C1498 cells and survival of recipient PD-1H KO mice was compared to WT controls. We observed that murine AML cell growth in vivo was diminished in PD-1H KO mice (mean radiance in one representative experiment: 2.6x10⁶ (KO) vs 4.2x10⁷ (WT) on day 26, N=5, p<0.05) and survival of PD-1H KO mice was improved compared to WT controls (median survival: 35 days (KO) vs 28 days (WT), N=5, p<0.05). In addition, PD-1H antibody (Ab) decreased AML cell growth in vivo in KO mice (mean radiance in one representative experiment: 2.4x10⁵ (KO + PD-1H Ab) vs 2.6x10⁶ (KO + Isotype control) on day 26, N=5, p<0.05) and extended survival in C1498-bearing PD-1H KO mice (median survival: 45 days (KO + PD-1H Ab) vs 35 days (KO + Isotype), N=5, p<0.05). These data suggest that PD-1H on both host cells and AML cells induces immune evasion in AML. Based on prior preclinical models, we further hypothesized that immunogenicity of AML can be enhanced by epigenetic modulation, that can potentiate anti-leukemic effect of PD-1H blockade. We found that DNA methyltransferase inhibition (DNMTi) by 5-aza-2'-deoxycytidine (decitabine) increased T cell infiltration in subcutaneously injected murine AML (C1498) tumor (CD3+ cells/tumor cells: 33±5% (DNMTi) vs 10±3% (PBS control), N=3, p<0.05) using flow cytometry and immunohistochemistry. Importantly, DNMTi potentiated the anti-leukemic effect of the PD-1H blockade (mean radiance in one representative experiment: 9.9x10⁸ (PBS + WT) vs 1.2x10⁸ (PBS + KO) vs 3.6x10⁵ (decitabine + WT) vs 6.9x10⁴ (decitabine + KO) on day 21; 5.9x10⁹ (PBS + WT) vs 3.5x10⁸ (PBS + KO) vs 1.8x10⁶ (decitabine + WT) vs 1.3x10⁵ (decitabine + KO) on day 24, N=7, p<0.05) and significantly prolonged survival in PD-1H KO mice (median survival: 28 days (WT) vs 35 days (KO) vs 39 days (DNMTi + WT) vs 60 days (DNMTi + KO), p<0.05). Together, our data suggest that PD-1H is highly expressed in human AML and can induce immune evasion in a murine model of AML (C1498). DNMTi has potential synergistic effects on PD-1H blockade and combining these two can be an important immunotherapeutic approach in AML.